线粒体毒物兴奋效应和(或)非折叠蛋白反应在Pink1敲低挽救SCA3/MJD转基因果蝇中的作用

Low-level oxidative stress induces an adaptive response commonly defined as hormesis; this type of stress is often related to reactive oxygen species (ROS) originating from the mitochondrial respiratory chain (mitochondrial hormesis or mitohormesis). The mitochondrial unfolded protein response is a conserved pathway that allows mitochondrial chaperones and other factors to be induced in response to mitochondrial dysfunction.PD-associated gene, PINK1, has been implicated in controlling mitochondrial morphology though increases fission and inhibits fusion. The latest evidences showed that down-expression of PINK1 caused mitochondrial elongation and deteriorate neurodegeneration of PD transgenic flies. Interestingly, in previous work we have shown that knockdown of pink1 by RNA interference (RNAi) significantly suppressed phenotype of Drosophila model of SCA3/MJD, Conversely, Knockout of PINK1 led to an enhanced neurodegenerative phenotype. In this study, we will utilize transgenic flies that mitochondrials of motor neuron were labeled by GFP fluorescence to definite that relationship between PINK1 knockdown significantly inhibits phenotype of SCA3/MJD transgenic flies, with different methods including RNA intervention, Western blotting and Real Time PCR. It is important to determinate whether mitohormesis or UPRmt contributes to the pink1 knock down rescuing SCA3/MJD fly. These results will provide strong support for therapy of SCA3/MJD through mitochondrial stress.

线粒体毒物兴奋效应（mitohormesis）和非折叠蛋白反应(UPRmt)是线粒体在低毒威胁下通过超补偿效应自我保护的两种应激机制，前者与活性氧(ROS)短暂增加介导的Foxo激活有关，后者与线粒体/核编码的氧化磷酸化蛋白失衡所诱导的线粒体分子伴侣核转录增加有关。我们在前期工作中发现，敲除线粒体功能基因Pink1显著加重了SCA3/MJD果蝇的神经变性，但其敲低反而提高了线粒体功能，挽救了果蝇表型。本研究拟通过qRT-PCR，RNA干扰，免疫印迹等方法，研究清除ROS或敲除Foxo是否能抑制Pink1敲低对SCA3/MJD果蝇的挽救作用，以及Pink1敲低是否破坏了果蝇神经元线粒体/核蛋白平衡，增加了hsp60等分子伴侣的表达，明确mitohormesis和（或）UPRmt在Pink1敲低挽救SCA3/MJD转基因果蝇中的作用，为利用适度线粒体应激治疗SCA3/MJD疾病提供理论依据。

研究中，我们证明了PINK1基因的敲除（Knock out）会加重SCA3/MJD转基因果蝇的神经变性，严重损害他们的线粒体功能，降低他们的飞行能力，缩短他们的寿命。然而PINK1的RNAi敲低（knockdown by RNA Interference）则挽救了SCA3/MJD转基因果蝇，并显著提高了它的线粒体功能。实验表明，无论是通过清除ROS（抗氧化）或敲除诱导mitohormesis所必需的Foxo基因都不能抑制Pink1敲低对SCA3/MJD转基因果蝇的保护作用，而将UPRmt效应标志物hsp60、ClpP以及自噬基因atg5等敲除后，其保护作用明显减弱，说明Pink1 敲低对SCA3/MJD 转基因果蝇表型的挽救作用与UPRmt相关，并且其保护作用可能与通过诱导自噬途径清除致病蛋白相关；同时本实验还证实了SCA3致病蛋白ataxin-3可以通过改善线粒体形态结构及功能保护PINK1敲除的PD转基因果蝇模型。